title: Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step.
abstract: Efficient labeling of protein-based targeting ligands with various cargos (drugs, imaging agents, nanoparticles, etc.) is essential to the fields of molecular imaging and targeted therapeutics. Many common bioconjugation techniques, however, are inefficient, nonstoichiometric, not site-specific, and/or incompatible with certain classes of protein scaffolds. Additionally, these techniques can result in a mixture of conjugated and unconjugated products, which are often difficult to separate. In this study, a bacterial sortase enzyme was utilized to condense targeting ligand purification and site-specific conjugation at the C-terminus into a single step. A model was produced to determine optimal reaction conditions for high conjugate purity and efficient utilization of cargo. As proof-of-principle, the sortase-tag expressed protein ligation (STEPL) technique was used to generate tumor-specific affinity ligands with fluorescent labels and/or azide modifications at high purity (>95%) such that it was not necessary to remove unconjugated impurities. Click chemistry was then used for the highly efficient and site-specific attachment of the azide-modified targeting ligands onto nanoparticles.
authors.affiliation: Department of Bioengineering, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Warden-Rothman
authors.forename: Ilaria
authors.initials: I
authors.lastname: Caturegli
authors.forename: Vladimir
authors.initials: V
authors.lastname: Popik
authors.forename: Andrew
authors.initials: A
authors.lastname: Tsourkas
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry*; D000818:Animals; D001386:Azides / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D015153:Blotting, Western; D057930:Click Chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008279:Magnetic Resonance Imaging / Q000379:methods*; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D010446:Peptide Fragments / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism
chemical_list: D019881:Aminoacyltransferases; D001386:Azides; D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases; D049452:Green Fluorescent Proteins; D010446:Peptide Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; C485184:enhanced green fluorescent protein; C120542:sortase A